Gypsy moth virus with enhanced polyhedra production stability

ABSTRACT

A virus having the identifying characteristics of ATCC VR2396 is disclosed. This virus has the trait of enhanced polyhedra production stability and resists forming a few polyhedra mutant virus. A method of protecting crops from insects comprising applying an insecticidally effective amount of virus having the identifying characteristics of ATCC VR2396 is also disclosed.

This is a continuation of application Ser. No. 08/015,961 filed Feb. 8,1993, now abandoned.

FIELD OF THE INVENTION

The general field of the present invention is methods of controllingplant pests, such as gypsy moths. Specifically, the field of the presentinvention is gypsy moth virus strains.

BACKGROUND

Chemical pesticides and fungicides are the most commonly used controlagents for forest insect pests and fungal diseases. In excess of 350billion pounds of these agents are used annually in the United States tocontrol pests and diseases in forestry, agriculture, and residentialareas. Broad spectrum insecticides and fungicides have adverse impactsnot only on their target organisms but also on beneficial insects andfungi and, consequently, on the entire ecosystem. In addition, chemicalresidues may cause health problems among the human population.

Interest in biological insect and fungal control agents is growing as aconsequence of concerns regarding chemical pesticide use. A number ofbacteria synthesize antifungal compounds and over 1500 microorganism andmicrobial products have been identified that are insecticidal.Generally, natural control agents have little adverse ecological impactdue to their specificity for the target host. Long term environmentalhazards and health concerns are not a factor with biological controlagents because chemical residues are not present. Unfortunately,biological control agents suffer from several disadvantages incomparison to chemical pesticides, including cost of production,efficacy, and stability.

One particularly troublesome plant pest is the gypsy moth Lymantriadispar. The gypsy moth was first imported from Europe into North Americanear Boston in 1869. Since then, the area of gypsy moth infestation hasincreased to include almost the entire New England area, New York,Delaware, Maryland, New Jersey, Pennsylvania, Virginia, West Virginia,Ohio, and Michigan. (McFadden, et al., Forest Insect Guilds: Patterns ofInteraction with Host Trees, Y. N. Baranchikov, et al. pp. 172-186. USDepartment of Agriculture, For. Serv. Gen. Tech. Rep. NE-153, Radnor,Pa., 1989.) Several chemical insecticides have been used for gypsy mothcontrol. DDT is one of the most effective chemical insecticides usedagainst gypsy moths.

Knowledge of the environmental impacts of DDT and other chemicalinsecticides has caused an industry shift to the use of agents such asDimilin and Bacillus thuringiensis (Bt) for gypsy moth control. TheLymantria dispar nuclear polyhedrosis virus (LdNPV), which is pathogenicto Lymantria dispar (gypsy moth), is also used as a biocontrol agent.LdNPV has the significant advantage over other control agents ofspecificity for the gypsy moth. Consequently, LdNPV is the agent ofchoice for all areas and particularly for use in environmentallysensitive areas. However, LdNPV is not used extensively for gypsy mothcontrol because of high production costs and low efficacy. Oneparticular problem with LdNPV propagation in cell culture is thepropensity of the virus to mutate into a form that produces fewerpolyhedra (FP mutants).

For gypsy moth virus production to be competitive, means of reducingproduction costs must be devised. One approach is the development ofinsect cell lines that produce high amounts of polyhedra. Lynn et al.,(Appl. Envron. Microbiol., 55, 1049-1051, 1989) have developed afat-body cell line that produces greater amounts of polyhedra than othergypsy moth cell lines.

What is needed in the art of gypsy moth control is an improved strain ofLdNPV capable of enhanced production.

SUMMARY OF THE INVENTION

The present invention is a virus having the identifying characteristicsof ATCC VR2396. This virus has the trait of enhanced resistant tomutation to an FP form of LdNPV virus.

The present invention is also a virus derived from ATCC VR2396, whereinthe virus retains the characteristics of polyhedra production stability.

The present invention is also a method of protecting plants frominsects, comprising applying to an insect or an insect habitat aninsecticidally effective amount of a virus having the identifyingcharacteristics of ATCC VR2396. In a preferred form of the invention,the insects are gypsy moths.

The present invention is also an insecticidal composition comprising aninsecticidal amount of a virus having the identifying characteristics ofATCC VR2396 and an inert carrier.

It is an object of the present invention to control insect pests.

It is another object of the present invention to provide an LdNPV viruswith enhanced polyhedra production stability.

It is another object of the present invention to provide an LdNPV viruswith enhanced resistance to mutating to an FP form of LdNPV virus.

It is an advantage of the present invention that insect control may beprovided at lower cost.

It is another advantage of the present invention that ATCC VR2396 may bepropagated with a fewer amount of FP mutants forming.

Other objects, advantages and features of the present invention willbecome apparent after review of the specification, drawings and claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graphical comparison of the amount of polyhedra produced incells infected with A21-MPV after 14 serial passages versus cellsinfected with A21-2 after 6 serial passages in L. dispar 652Y cells;FIG. 1B is a tabular form of the same data. The values in FIG. 1B arethe means of three determinations ± the standard deviation of the numberof polyhedra per cell of cells containing polyhedra after infection with0.2 TCID/50 units per cell.

FIG. 2A is a graphical comparison of the number of cells found tocontain polyhedra after infection with A21-MPV after 14 serial passagesversus the number of cells found to contain polyhedra after infectionwith A21-2 after 6 serial passages in L. dispar 652Y cells; FIG. 2B is atabular form of the same data. The values in FIG. 2B are the means ofthree determinations ± the standard deviation of the percentage of cellscontaining polyhedra after infection with 0.2 TCID/50 units per cell.

FIG. 3A is a graphical comparison of the amount of budded virus producedby isolates A21-MPV after 14 serial passages and A21-2 after 6 serialpassages in L. dispar cells;

FIG. 3B is a tabular form of the same data. The values in FIG. 3B arethe means of three determinations ± the standard deviation of the buddedvirus TCID/50 values per ml. of media after infection with 0.2 TCID/50units per cell.

FIG. 4A demonstrates the retention of wild-type phenotype by A21-MPVthrough multiple passages;

FIG. 4B demonstrates the percentage of cells with polyhedra afterinfection with A21-MPV during extended serial passage in L. dispar 652Ycells.

FIG. 5A is a graphical comparison of polyhedra per cell of cellsinfected with A21-MPV and cells infected with A21 during serial passage;

FIG. 5B is a tabular form of the same data. The values in FIG. 5B arethe means of three determinations ± the standard deviation of the numberof polyhedra per cell of cells containing polyhedra 7 days after viralinfection with 0.2 TCID/50 units per cell. The means within a columnfollowed by different letters are significantly different at less thanthe 0.05 level.

FIG. 6A is a graphical comparison of the percentage of polyhedra incells infected with A21-MPV versus A21 during serial passage;

FIG. 6B is a tabular form of the same data. The values in FIG. 6B arethe means of three determinations ± the standard deviation of thepercentage of cells containing polyhedra 7 days after infection with 0.2TCID/50 units per cell. The means within columns followed by differentletters are significantly different at <0.05 level.

FIG. 7A is a graphical comparison of budded virus produced by isolatesA21 and A21-MPV during serial passage in L. dispar 652Y cells;

FIG. 7B is a tabular form of the same data. The values in FIG. 7B arethe means of three determinations ± the standard deviation of the buddedvirus TCID/50 values per ml. of media 7 days after infection with 0.2TCID/50 units per cell. The means within columns followed by differentletters are significantly different at <0.05 level.

FIG. 8 indicates the percentage of A21 plaques exhibiting the FPphenotype compared to the number of A21MPV plaques exhibiting the FPphenotype during serial passages.

DESCRIPTION OF THE INVENTION

1. Brief Overview

We have identified an LdNPV isolate, derived from LdNPV isolate A21,that exhibits an enhanced stability of wild-type polyhedra productionlevels during passage in L. dispar 652Y cells. All wild-type LdNPVisolates studied to date rapidly mutate during serial passage in 652Ycells to few polyhedra (FP) viral mutants. FP mutants exhibit thecharacteristics of production of an average of 4-10 polyhedra per cell,of polyhedra that are virtually devoid of viral nucleocapsids, and of agreater amount of budded virus compared to wild-type virus. In contrast,wild-type virus produce an average of 30-60 polyhedra per cell thatcontain many viral nucleocapsids and a lower amount of budded viruscompared to FP mutants.

The unique attribute of our new isolate, A21-MPV, is a reduced tendencyto mutate to an FP mutant during serial passage. Consequently, duringproduction of LdNPV polyhedra, which requires serial passage, isolateA21-MPV will produce a far greater number of polyhedra than wild-typevirus. In addition, a greater percentage of 652Y cells infected withisolate A21-MPV produce polyhedra in comparison to the percentage ofcells that produce polyhedra after infection with wild-type LdNPV.

2. In General

Two forms of LdNPV (occluded and non-occluded virus) are produced duringviral replication. (Granados, et al., The Biology of Baculoviruses, R.R. Granados and B. A. Federici (eds.) pp. 89-108. CRC Press, Boca Raton,Fla., 1986.) Early after viral infection, non-occluded virus isproduced. This virus leaves the cell and is responsible for secondaryinfections within the hemocoel of the host insect. A different form ofvirus is produced late in infection that is occluded into a proteinmatrix composed primarily of a viral-encoded protein. These structures,approximately 1 to 3 micrometers in diameter and polyhedral in shape,are termed "polyhedral occlusion bodies" or "polyhedra". Nucleocapsidswithin polyhedra are protected from most environmental conditions withthe exception of ultraviolet light.

Gypsy moth virus is applied through sprayers in the field in the form ofpolyhedra, which are ingested by gypsy moth larvae feeding on treefoliage. Once within the alkaline environment of the insect midgut, thepolyhedra dissolve and release nucleocapsids which infect the insectmidgut cells, thereby initiating the infection process.

For commercial applications, LdNPV is currently produced in gypsy mothlarvae at a cost of approximately $30.00 for enough polyhedra to treatan acre of forest. Production is the most expensive component in thecommercial use of LdNPV. Virus production is limited by the efficiencyof production of polyhedra in larvae. Efforts to produce polyhedra incell culture systems are being made to generate a more economicalproduction methodology. In comparison, production costs of Dimilin andBt are approximately $3.00 per acre equivalent.

However, problems exist in obtaining virus from cell culture. A class ofmutant baculoviruses called few polyhedra (FP) mutants are generated athigh frequency during viral replication in insect cell culture. FPmutants have the characteristics of producing few polyhedra that containfew or no viral nucleocapsids and are non-infectious. In addition, FPmutants produce a greater amount of budded virus (non-occluded virus)compared to wild type virus.

FP mutants have been generated during serial passage of the Autographacalifornica nuclear polyhdrosis virus (AcMNPV), Trichoplusia pi MNPV(TnMNPV), Galleria mellonella MNPV (GmMNPV), and Lymantria dispar MNPV.Studies on AcMNPV have shown that FP mutants are often generated throughthe insertion of host DNA sequences into the viral genome. The frequencyof FP mutant genesis during viral serial passage is specific to thevirus species being passed. Table 1 below summarizes the frequency of FPmutant formation as a function of serial passage.

                  TABLE 1                                                         ______________________________________                                        Passages Necessary to Accumulate FP                                           Mutant Percentages of:                                                        Virus        25%    50%        75%  >90%                                      ______________________________________                                        GmNMPV.sup.a 4.5    5.5        7.0   8.0                                      TnMNPV.sup.b 4.0    5.0        6.0  10.0                                      AcMNPV.sup.c 1.0    1.5        3.5  >4.0                                      LdMNPV.sup.d        1.0              2.0                                      ______________________________________                                         .sup.a. Fraser, et al., (1982) Virology, 117, 366-378.                        .sup.b. Potter, et al., (1976) J. Virology, 18, 1040-1050.                    .sup.c. Hink, et al., (1976) J. Invertebrate Pathology, 27, 49-55.            .sup.d. Slavicek, J. M. Unpublished data.                                

Our approach to increase polyhedra production in cell culture is thedevelopment of a viral strain that exhibits a low frequency of FP mutantgeneration and consistently produces polyhedra without reductions as aconsequence of passage. These traits are what we mean by "polyhedraproduction stability".

Our preliminary studies with LdNPV polyhedra production in 652Y cellsfound a reduction in the number of polyhedra produced as a function ofvirus passage number. When non-occluded virus is obtained fromfourth-instar gypsy moth larvae (that were infected per os withpolyhedra) and used to infect 652Y cells, the infected cells produceapproximately 50 polyhedra per cell (Table 2). After five passages incell culture, the number of polyhedra produced per cell decreased toapproximately 6. The cost of cell culture production could be reduced ifthe initial high level of polyhedra formation could be stabilized.

We have identified a viral variant (termed A21-MPV, which stands forMany Polyhedra Variant) that maintains a high level of polyhedrasynthesis after repeated passage in cell culture (Table 2). 652Y cellsinfected with MPV contain approximately 45 polyhedra per cell. Inaddition, more of the infected cells produce polyhedra in comparison towild type isolate A21. Virus A21-MPV has been deposited with theAmerican Type Culture Collection, Rockville, Md., on Dec. 16, 1992 atAccession Number VR2396 under the conditions of the Budapest Treaty.Deposit of this sample does not imply or grant a license to use thevirus.

                  TABLE 2                                                         ______________________________________                                                           Polyhedra                                                          1st passage                                                                              production per                                                                             Percentage of                                         polyhedra  cell after more                                                                            cells containing                              Isolate production than 5 passages                                                                            polyhedra                                     ______________________________________                                        A21     52          6           51                                            A21-MPV 58         45           85                                            ______________________________________                                    

3. A21-MPV Isolation

LdNPV isolates A21, B21, 122, and 163 were purified to genotypichomogeneity by low dose infection of L. dispar larvae. Fourth instarlarvae were fed diet containing a dose of LdNPV that causedapproximately 4 to 10% mortality. Polyhedra were collected fromindividual cadavers, and used for a second round of low dose infections.Polyhedra were collected from individual cadavers, viral genomic DNA wasisolated and analyzed to assess the degree of genotypic heterogeneitypresent. Isolates A21, B21, 163 and 122 appeared to be genotypicallyhomogenous and were used for further investigations.

The initial purpose of the experiment during which we isolated A21-MPVwas to develop the methodology for generation of recombinant viruses.LdMNPV isolate A21 and a transplacement plasmid pLd-B-Gal that containsthe beta galactosidase gene from E. coli were examined in this study.

Lymantria dispar 652Y cells were propagated in modified Goodwin'sIPL-52B medium with 10% heat-inactivated fetal bovine serum and 6.25 mMglutamine. 2×10⁵ cells were plated per well in P6 plates (Corning) priorto transfection. Media was removed and replaced with 1.5 ml serum freemedia, and 100 ul of lipofection solution (50 ul 10 mM Tris, 1 mM EDTA,pH 7.5; 2 ug, 5 ug, and 10 ug of LdMNPV DNA, and 4 ug, 2 ug, and 4 ug ofpLd-B-Gal DNA for trials A, B, and C, respectively; and 50 ul Lipofectinreagent, BRL) was added to the P6 wells. After a 4 hour incubationperiod the media was replaced with complete media containing 50 ug/mlgentamicin. Fifteen days after transfection the cells were harvested andused for enzymatic B-galactosidase assays to detect the presence ofrecombinant LdMNPV containing the B-galactosidase gene. The percentageof recombinant virus present was calculated after determination ofrecombinant virus titer by plaque assay (using agarose containing BluoGal supplied by Gibco) and the total virus titer by TCID-50 Assay.

LdMNPV isolate A21-MPV was identified in this study. However, A21-MPVdoes not contain the beta-galactosidase gene. Isolate A21-MPV is amutant derivative of isolate A21 that was detected during the course ofthe study described above. The genesis of A21-MPV is not related to theexperimental manipulations used to generate a recombinant virus. Cellscontaining isolate A21-MPV were recognized through examination ofinfected cells by light microscopy. These cells were black in color, incontrast to all other cells infected with isolate A21, due to the largenumber of polyhedra present. The majority of infected cells, greaterthan 99.99%, contained from 4 to 10 polyhedra, which is characteristicof FP viral mutants. The virus that infected the cells containing alarge number of polyhedra was purified and termed LdNPV isolate A21-MPV(many polyhedra variant, MPV). Isolate A21-MPV was serially passed incell culture for 20 passages. The number of 652Y cells used for eachpassage were 1×10⁴, 1×10⁵, 1×10⁶, and 5×10⁶ for passages 1-8, 9-10, 11and 12-20, respectively.

To generate cell culture lines of LdNPV isolates A21, B21, 163, and 122,budded virus was obtained from L. dispar larvae infected with theseisolates and used to infect 652Y cells in culture. Budded virus fromthese initial infections was used to generate the plaque purified virallines A21-2, B21-1, 122-2, and 163-2. After plaque purification theisolates were serially passed in cell culture for 6 passages. The numberof 652Y cells infected at each passage were 1×10⁴, 1×10⁵, 1×10⁶, and5×10⁶ for passages 1, 2, 3 and 4-6, respectively.

4. Restriction endonuclease analysis of isolate A21-MPV.

A21 and A21-MPV genomes were subject to restriction endonucleasedigestion with BglII, EcoRI, EcoRV, HindIII, BamHI, and PstI, and thedigestion profiles were compared to determine whether DNA restrictionfragment length polymorphisms existed between the viruses. Nodifferences in DNA fragment lengths were detected. This finding supportsthe hypothesis that isolate A21-MPV is a mutant derivative of isolateA21. The approach used in this comparison will detect DNA insertions ordeletions of approximately 0.5 kb or greater in length, and nucleotideadditions, deletions or alterations of the restriction endonucleaserecognition sites of the enzymes used.

5. Bioassay data on isolate A21-MPV, A21, and Gypchek.

Comparative bioassays were performed as described in Slavicek et al.,(1992, J. Invertebrate Pathology 59: 142-148). Briefly, polyhedra pastesof the various isolates were suspended in 10 ml of Sterile 0.05 MTris-HCl buffer, pH 7.2, and briefly sonicated to disperse thepolyhedra. A 100-mg lyophilized sample of standard gypsy moth virus,Gypchek, was similarly suspended and vigorously ground in a glass tissuehomogenizer for 3 min. Five serial 10-fold dilutions of each of thestock virus suspensions were prepared in Tris buffer. Dilutions wereprepared such that final polyhedra concentrations in diet fed to testinsects ranged between 1×10² and 1×10⁷ /ml. One milliliter of dilutionwas added to 99 ml of tempered (52° C.) diet and vigorously mixed with avariable-speed stirrer (5000 rpm) for 30 sec. Virus-treated diet wascooled and cooled and cut into 1.25-cm³ cubes.

Each virus dosage was presented to five groups of 10 newly molted secondinstar larvae from a standard laboratory strain of the gypsy moth (NewJersey, F₃₄). Each group of 10 larvae was confined to a 100×15-mmplastic petri dish and given two 1.25-cm³ virus-treated diet cubes onwhich to feed for 48 hr. Larvae were given untreated diet ad libitum forthe remaining 12 days of the bioassay. Five groups of 10 larvae(controls) were fed untreated diet for 14 days. Larvae were reared in agrowth chamber at 24°±2° under a 16/8-hr light/dark photoperiod andobserved daily for mortality. Dead larvae were removed and examinedmicroscopically to confirm MPV deaths.

Mortality data were examined by Probit analysis using a POLO-PC program(LeOra Software, Berkeley, Calif.). Potencies of isolates A21-MPV andA21 relative to standard Gypchek were estimated by comparing LC₅₀values.

Gypchek is the name of the LdMNPV registered by the Forest Service andis used currently for gypsy moth control. Gypchek is available from U.S.Forest Service, Northeastern Forest Experiment Station, Hamden, Conn.

The results of the bioassay are tabulated in Table 3.

                  TABLE 3                                                         ______________________________________                                        Isolate LC-50.sup.a                                                                            Limits.sup.b                                                                             LC-90.sup.a                                                                          Limits                                     ______________________________________                                        Gypchek 3800     2500-6000  24,000 14,000-56,000                              A21     4700     2800-7800  32,000 17,000-82,000                              A21-MPV 5000     3400-7600  39,000 23,000-86,000                              ______________________________________                                         .sup.a. LC 50 and LC90 (lethal concentration) values are the number of        polyhedra per ml of diet that cause 50% and 90% larval mortality,             respectively.                                                                 .sup.b. Limit values are numbers of polyhedra per ml of diet that cause       50% and 90% larvae mortality, respectively.                              

The minor differences found in LC-50 and LC-90 values exhibited byisolates A21, A21-MPV and Gypchek were not significant. This findingindicates that the biological activity of A21-MPV is essentially thesame as Gypchek, and consequently A21-MPV could be used for control ofthe gypsy moth.

6. Microscopy Comparison

Comparison of L. dispar 652Y cells infected with LdMNPV isolates A21-2and A21-MPV via light microscopy demonstrates the difference inpolyhedra production and, thus, the biological difference between thetwo viruses. 652Y cells were infected with 0.2 TCID-50 units per cell ofA21-2 and A21-MPV. Seven days after infection the cells were examined bylight microscopy and photographed at 100X magnification. The cellsinfected with isolate A21-2 contain very few polyhedra. In contrast,cells infected with A21-MPV contain a large number of polyhedra andappear black at this magnification level.

Electron micrographs of polyhedra cross-sections produced by isolate A21and A21-MPV infection at passage 1 and passage 5 can also differentiatebetween the two strains. 652Y cells were infected with 0.2 TCID-50 unitsper cell of either A21 or A21-MPV. Seven days after infection polyhedrawere harvested and prepared for examination by electron microscopy.

To prepare for electron microscopy, polyhedra were fixed in sodiumcacodylate buffer, pH 7.2 (0.05 M sodium cacodylate, 0.5 mM HCl),containing 1% glutaraldehyde at 4° C. for 20 min. Polyhedra were thenfixed in sodium cacodylate buffer with 3% glutaraldehyde for 1 hr at 4°C. The samples were washed 3X in sodium cacodylate buffer over a 1-hrperiod. The samples were postfixed in sodium cacodylate buffercontaining 2% osmium tetroxide for 2.5 hr at ambient temperature andthen washed 3X in sodium cacodylate buffer over a 1-hr period. Moltenagar was added to the samples (final concentration 2%) and allowed togel. The samples then were cut into 1-mm² blocks and incubated overnightin 35% ethanol. The samples were dehydrated through incubation in anethanol series (35, 50, and 70% with 2% uranylacetate, 85, 95, 100%-3X)for 5-7 min at each step, except for the 70% step which was for 1 hr.The samples were then washed 3X (10 min each wash) in propylene oxide.

The samples were infiltrated over a 24-hr period with propyleneoxide:Poly/Bed 812 (25.55 g poly bed, 13.5 g dodecenylsuccinicanhydride, 10.9 g nadic methylanhydride, Polysciences) at 2:1, 1:1, and1:2 ratios. After infiltration, the polyethylene capsules were flushedwith freon and filled with Poly/Bed 812 containing 2% DMP 30(Polysciences), incubated at 35° C. for approximately 15 hr, 45° C. for8 hr and finally at 60° C. overnight. The samples were sectioned with adiamond knife on a Reichert-Jung Ultracut E43 microtome, the sectionsstained for 20 min in 5% uranylacetate in methanol, poststained in leadnitrate (2.7% lead citrate, 3.5% sodium citrate, 0.16 N NaOH), andviewed with a Hitachi model HU 11E-1 transmission electron microscope.Polyhedra cross sections were photographed, and the number of virionspresent within cross-sections were quantified by counting.

In the A21 samples, numerous viral nucleocapsids are evident incross-sections produced at the first passage. In contrast,cross-sections of polyhedra produced at the fifth passage contain few orno viral nucleocapsids. In the A21-MPV sample, numerous viralnucleocapsids are evident in cross-sections produced at both the firstpassage and fifth passage. The presence of numerous viral nucleocapsidsin polyhedra produced at the fifth passage by A21-MPV indicates thatthis isolate is producing normal polyhedra at this passage, in contrastto isolate A21.

7. Comparison of Polyhedra Production Attributes of Isolates A21-MPV andA21-2 After Plaque Purification in Cell Culture.

The number of polyhedra produced per cell within cells containingpolyhedra, the percentage of cells with polyhedra, and the amount ofbudded virus produced by isolates A21-MPV after 14 passages in cellculture and isolate A21-2 after 6 passages in cell culture weredetermined and compared. The number of polyhedra per cell produced byisolates A21-MPV and A21-2 was determined 3, 5, and 7 days postinfection (p.i.). On all days p.i. examined, cells infected with isolateA21-MPV contained significantly more polyhedra per cell within cellscontaining polyhedra compared to cells infected with isolate A21-2 (FIG.1). At seven days p.i. cells infected with A21-MPV contained an averageof 46.4 polyhedra, in contrast to 5.2 polyhedra per cell in cellsinfected with A21-2.

The percentage of cells containing polyhedra was determined 3, 5, and 7days p.i. A significantly greater percentage of cells were found tocontain polyhedra at 3, 5, infected with A21-2 (FIG. 2). At 7 days p.i.an average of 85.7% of cells infected with A21-MPV contained polyhedra.In contrast, 64.3% of cells infected with A21-2 contained polyhedra 7days p.i.

The amount of budded virus produced by isolates A21-MPV and A21-2 wasdetermined 1, 3, 5, and 7 days p.i. On days 3, 5, and 7 p.i., A21-2produced a greater amount of budded virus in comparison to A21-MPV (FIG.3). On days 5 and 7 p.i. A21-2 produced significantly more budded virusthan A21-MPV.

These results demonstrate that isolate A21-2 exhibits the attributes ofan FP mutant at the 6th passage in cell culture. In contrast, A21-MPVhas retained a wild-type polyhedra production phenotype through the 14thserial passage. A21-MPV was then serially passed an additional 6 timesthrough passage 20. As shown in FIG. 4, A21-MPV retained a wild-typephenotype through the 20th passage. These results demonstrate theenhanced stability of polyhedra production exhibited by A21-MPV.

Isolates B21-1, 122-2 and 163-2 were serially passed in 652Y cells for 6passages as described for isolate A21-2. Similarly to A21-2 isolatesB21-1, 122-2, and 163-2 exhibited the attributes of FP mutants at the6th passage. This result demonstrates the high frequency of FP mutantsat the 6th passage and demonstrates the high frequency of FP mutantformation with wild-type strains of LdNPV in contrast to isolateA21-MPV.

8. Polyhedra Production Characteristics During Serial Passage

To further determine the stability of polyhedra production by A21-MPVand determine the number of passages necessary for a wild-type virus tomutate to an FP mutant, polyhedra production by isolates A21 and A21-MPVwere examined through five passages in cell culture. Viral nucleocapsidocclusion characteristics of A21 and A21-MPV during serial passage arerelated below in Table 4. LdMNPV isolates A21 and A21-MPV were seriallypassed five times in L. dispar 652Y. L. dispar larvae were infected peros with A21 (non-plaque purified) polyhedra produced in larvae andA21-MPV polyhedra produced in cell culture. Five days after infectionlarvae were bled, and the hemolymph was used to infect 1×10⁶ L. dispar652Y cells at 0.2 TCID/50 units per cell. This first infection in cellculture was passage number 1. Budded virus from this infection was thenused to infect new cells to generate passage 2. The remaining passagesexamined in this study, passages 3-5, were generated using budded virusfrom the previous passage as inoculum.

Polyhedra produced by these isolates after each passage were preparedfor examination by electron microscopy as described above. The number ofviral nucleocapsids present in polyhedra cross sections were determined,and are presented below in terms of the number of nucleocapsids presentper square micrometer of cross section surface area (Table 4).

                  TABLE 4                                                         ______________________________________                                        Number of viral nucleocapsids per um.sup.2 of cross section area                      Passage  Passage  Passage                                                                              Passage                                                                              Passage                               Isolate 1        2        3      4      5                                     ______________________________________                                        A21     5.7      4.0      2.5    1.1    0.5                                   A21-MPV 5.2      2.9      5.7    6.1    3.1                                   ______________________________________                                    

The number of viral nucleocapsids present in polyhedra produced by A21decreased during serial passage from an average of 5.7 at passage 1 to0.5 at passage 5. In contrast, the number of nucleocapsids present inpolyhedra produced by isolate A21-MPV remained essentially constantduring five serial passages.

L. dispar 652Y cells exhibited essentially the same number of polyhedraper cell at the first passage infection with isolates A21-MPV and A21(FIG. 5). In contrast, at passages two through five, cells infected withA21-MPV contained significantly more polyhedra per cell compared tocells infected with A21 (FIG. 5). After five serial passages, cellsinfected with A21-MPV contained an average of 35.4 polyhedra. Incontrast, cells infected with A21 contained an average of only 6.4polyhedra per cell. After each serial passage, cells infected with A21contained significantly fewer polyhedra in comparison to the previouspassage. In contrast, no significant difference was found in the numberof polyhedra per cell after infection with A21-MPV during five serialpassages.

At the first passage, cells infected with A21-MPV and A21 exhibited thesame percentage of cells containing polyhedra. At passages two throughfive a significantly greater percentage of cells infected with A21-MPVcontained polyhedra in comparison to cells infected with A21. At passagefive, 84.0% of cells infected with A21-MPV contained polyhedra whereasonly 51.2% of cells infected with A21 contained polyhedra. In addition,A21-MPV exhibited a significant increase in the percentage of cellscontaining polyhedra after the first passage. In contrast, A21 exhibiteda significant reduction in the percentage of cells containing polyhedraafter the first passage. FIG. 6 graphically displays these results.

Isolate A21-MPV exhibited stable budded virus production during fiveserial passages. The amount of budded virus produced by A21-MPV was lessthan the amount of budded virus produced by A21 at passages 2-5. Thisdifference was found to be significant at passages 2, 4, and 5, andnearly significant at passage 3. In addition, A21 exhibited an increasein budded virus production after the first passage. FIG. 7 graphicallydisplays these results.

The percentage of A21-MPV and A21 viruses exhibiting the FP or MPphenotype was determined during serial passage. Budded virus produced ateach serial passage was used to generate viral plaques. Ninety-sixplaques of each isolate after each passage were used to infect L. dispar652Y cells, and the infections were scored as exhibiting either the MPor FP phenotype. Budded virus obtained from insect hemolymph was usedfor passage zero for both viral isolates. As shown in FIG. 8, thepercentage of A21 plaques exhibiting the FP phenotype rapidly increasedduring serial passage from zero to 82% after just two serial passages.In contrast, A21-MPV maintained the MP phenotype during cell passage.

9. Viral formulation and application for gypsy moth control.

We envision that the A21-MPV isolate may be applied to insects or insecthabitats in a manner similar as that currently used for LdMNPV. Thevirus must be ingested by the insect to be effective. However, insectsmay also become infected as a consequence of ingestion of virus presenton the insect or on insect egg masses. Therefore, in the case of plants,the virus will typically be applied to leaf surfaces.

LdMNPV has been formulated as described below for gypsy moth control:

10 g Gypchek (5.0×10¹⁰ polyhedra per gram)

227 g (6% wt/vol) Orzan LS (a sunscreen, ITT Raynonier, Seattle, Wash.)

0.47 liters (12.5% by vol) Pro Mo liquid supplement (Southern StatesCooperative, Richmond, Va.)

77.6 ml (2% by vol) Rhoplex B60A (a sticker-spreader, Rohm & HaasCompany, Philadelphia, Pa.) 3.24 liters (85.5% by vol) water

A similar preparation is envisioned for the A21-MPV isolate.

The amount of virus to be administered to the insect or the insecthabitat is an amount effective to reduce insect infestation aspredetermined by routine testing. If the ultimate response is insectmortality, an "insecticidally effective amount" is defined to be thosequalities of virus which will result in a significant mortality rate ofa test group, as compared to an untreated group. The insecticidallyeffective amount may vary with the species of pest, stage of larvaldevelopment, nature of the substrate, the type of vehicle or carrier,the period of treatment and other factors.

It is often advantageous to apply viral inoculants with a carrier.Suitable agronomically acceptable carriers are known in the art. Inertsolids, such as cellulose or sugars, wetable powders, and aqueoussurfactant mixtures are illustrative of suitable chemical carriers.Depending on many factors, the concentration of virus in the finalcomposition may vary, but would include an insecticidally effectiveamount which may typically be between 1×10⁸ to 1×10¹¹ polyhedra perliter, but the preferred embodiment would be between 1×10¹⁰ to 1×10¹¹polyhedra per liter.

Aerial application of Gypchek has been as follows: A 448 kW (600 hp)Grumman Agcat airplane equipped with 8 Micronair AU 5000 Atomizers canbe used for LdNMPV application. Delivery of formulated virus could befrom 1.0×10¹¹ to 5.0×10¹¹ polyhedra in 7.5 liters per acre at anairspeed of 160 km/h. (Podgwaite, et al., J. Economic Entomology, 85:1136-1139).

We claim:
 1. A virus designated ATCC VR2396 deposited at the AmericanType Culture Collection.
 2. An insecticidal composition comprising:aninsecticidal amount of the virus of claim 1 and an inert carrier.